改进与核移植方法完成小鼠卵丘细胞重组胚与早期发育.docVIP

改进的核移植方法完成小鼠卵丘细胞重组胚的早期发育 作者：沈新明1,2；乔贵林1；江培洲1；李欣1；黄华1；姚开泰1　　(1南方医科大学肿瘤研究所，广东??广州??510515，2上海市东方肝胆外科医院，上海??200438) 摘要：目的??建立一种快速、简便、有效且损伤小的核移植方法，研究来源于小鼠体细胞重组胚的早期发育。方法??用尖锐的去核针在MⅡ期卵母细胞透明带上切开一个25 mm左右的小口，首先将第一极体挑出，再轻轻地挤压和负压吸引卵母细胞，去除包含有MⅡ期染色体-纺锤体复合体的最少量细胞质。随后，将直径为10~12 mm的C57BL/6j小鼠卵丘细胞核注射到去核卵母细胞透明带下，构建供体核-卵母细胞复合体。用电融合的方法诱导复合体融合，并对重组胚激活。体外培养72 h，对重组胚发育到2细胞、4~8细胞和桑椹胚期等各阶段进行观察和计数。结果??完成一个MⅡ期卵母细胞去核的平均时间为15 s；Hoechst 33342染色证明可以完全去除MⅡ期卵母细胞细胞核；去核成功率为95.5%，注核成功率为76.7%，供体核-卵母细胞复合体融合和重组胚原核形成率分别为68.2%和62.2%；重组胚体外培养72 h内发育到2细胞、4~8细胞和桑椹胚期的比率分别为57.5%、39.1%和27.6%；用2个微卫星位点D7Mit22和D4Mit204引物，扩增不同发育阶段重组胚的DNA片段，表明重组胚来源于供体卵丘细胞。结论??应用改进的核移植方法，不仅可以有效地去除卵母细胞细胞核，去核成功率高，而且操作简便，去核迅速，对卵母细胞损伤小，是研究小鼠体细胞核移植的一种简便可行的新方法。 关键词：细胞核/移植；电融合；卵母细胞；重组胚 中图分类号：R329.25??文献标识码：A??文章编号：1000-2588(2005)06-0613-06 A modified method of nuclear transfer for investigating early development of mouse embryos reconstructed with cumulus cell nucleiSHEN Xin-ming1,2； QIAO Gui-lin1； JIANG Pei-zhou1； LI Xin1； HUANG Hua1； YAO Kai-tai11Institute of Cancer Research, Southern Medical University, Guangzhou 510515, China; 2Eastern Hepatobiliary Hospital, Shanghai 200438, ChinaAbstract: Objective To establish a fast, simple, efficient and minimally invasive method for nuclear transfer (NT) to study the early development of mouse embryos reconstructed with cumulus cell nuclei in vitro. Methods With a sharp-tipped enucleation needle, an incision approximately 25 mm in length was made in the zona pellucida of a rat oocyte, through which the first polar body was removed and the metaphaseⅡchromosome-spindle complex was gently aspirated along with a minimal volume of the cytoplasm by slightly pressing and vacuum aspiration of the oocyte. A cumulus cell nuclei from C57BL/6j mouse, 10-12 mm in diameter, was inserted into the perivitelline space of the enucleated oocyte. The fusion of the donor-recipient pair was induced by electrofusion and nuclear formation was observed. The development of 2-cell, 4- to 8-cell and morula-stage embry